Ovarian cancer (OVCA) is the most lethal gynecologic malignancy. The majority of patients are diagnosed with advanced disease, which ultimately recurs, and they die from the disease. OVCA is becoming resistant to current chemotherapies, including the two most commonly used first-line drugs taxol and cisplatin, and patients are exhausting their treatment options. Multidrug resistance (MDR) is closely related to overexpression of membrane efflux proteins (e.g., P-glycoprotein) and anti-apoptotic proteins [e.g., survivin and myeloid cell leukemia 1 (Mcl-1)]. Because small RNA molecules, including small interfering RNA (siRNA), have an extraordinary ability to knock down gene expression, RNA interference (RNAi) induced by small RNA molecules can be an excellent solution for overcoming MDR. RNA molecules are highly susceptible for enzymatic degradation and too big to penetrate the cellular membranes. Although various types of delivery materials have been developed and used at the in vitro tissue culture level, gene regulation at the ex vivo or in vivo level has been largely unsuccessful due to poor intracellular siRNA availability. Lack of targeting and inefficient intracellular entry of drugs requires over dosing, which is also responsible for poor therapeutic outcomes. Efficient delivery of negatively charged RNA molecules to target cells is pivotal for a successful application of RNAi technology. Innovative therapeutic delivery techniques are urgently needed to address the drug resistance and poor intracellular entry efficiency.
In human airway epithelium possessing additional extracellular barriers, such as mucus layers, transfection using conventional lipid-based or positively charged carriers is extremely limited. As the critical physical barrier interfacing environmental stimuli, the mucosal surfaces of epithelium tightly regulate various physiological and immunological processes. In the mucus layer, dense mucin fibers and negatively charged proteoglycans provide the adhesive and viscous protective layer that often trap and remove positively charged carriers, resulting in poor delivery of payloads to the underlying epithelial cells. Very few options are currently available for delivering nucleic acids to the airway epithelium. Mucus-altering or mucolytic agents can be used as adjuvants of gene carriers, although high millimolar concentrations are often needed to disrupt or disturb the mucus layers.
Alternatively, block copolymers of polyethyleneimine (PEI) and polyethyleneglycol (PEG) have been developed to deliver plasmid DNA (pDNA) to the lung airways. Positively charged PEI and negatively charged pDNA form ionic complexes, while the PEG block shields the positively charged block from the negatively charged mucus layers and provides diffusion through nanometer sized mucus meshes. However, the PEG block often causes poor gene complexation and reduced cellular entry; and pDNA can form smaller ionic complexes with PEI-PEG copolymers due to the molecular topography of pDNA, enabling compaction to nanoparticles. Although optimization provides the opportunity to balance the ratios between charged and PEG segments, block copolymer architectures in biological fluids containing ions and proteins complicate surface properties and influence biological functions.
Short peptides or their synthetic mimics of the protein's translocation domains are excellent materials to introduce therapeutic agents to intracellular compartments rapidly. The fast entry of those materials is associated with a combination of membrane pore formation and non-receptor-mediated endocytosis. Combinations of ionic bonding, hydrogen bonding, and hydrophobic interactions influence the entry pathways. However, coupling cell penetrating peptides to therapeutic proteins or nucleic acids often alters the entry pathways, resulting in decreased intracellular availability. Fluorescent labels needed to study the entry mechanism and the localization of synthetic materials influences the materials physical properties and cellular behaviors toward the materials. The development of nontoxic biomaterials exhibiting superior cellular entry and therapeutic delivery is needed to substantially increase therapeutic efficacy of these systems.
In another approach, nanometer sized particles accumulate in relatively loosely organized tumor tissues as opposed to tight normal tissue. When the particulates are modified with ligands specific to the receptors overexpressed on cancer cell surfaces, targeting at the tissue level can be further improved. Unfortunately, overall therapeutic efficacy remains unsatisfactory due to poor intracellular entry and a lack of organelle targeting. Endocytosis mediated by cell surface receptors is the primary entry pathway, but it is often slow and inefficient. Endocytosed therapeutic agents undergo degradation in endosomes and lysosomes or in recycling processes, such as exocytosis, which lower the intracellular concentration of therapeutic agents. By not involving in endosome escaping process, direct membrane translocation offers high intracellular concentrations of therapeutics. Nanometer sized particles with modulated surface properties is pivotal for efficient intracellular delivery and labeling because the surface properties are closely related to their initial interaction following entry.
Aromatic π-electron conjugated polymers (CPs) are innovative fluorescent materials that have a high potential as therapeutic carriers. Because of excellent photophysical properties, such as high brightness and sensing ability, and excellent biophysical properties, such as biocompatibility, nontoxicity, high cellular interaction, and ease of entry, CPs have been used for live cell and tissue imaging, biochemical sensing, and gene and drug delivery. In addition to intrinsic fluorescent properties that are highly advantageous for labeling and tracking, the charged CPs are structurally similar in charge density and backbone rigidity to materials known for exhibiting efficient cellular entry, such as tyrosine aminotransferase (TAT) as shown in FIG. 1. Because of a rigid hydrophobic backbone and a flexible hydrophilic charged side chains, CPs can bind to and enter through cellular membranes.
Moon et al. U.S. Pat. Nos. 9,676,886 and 9,757,410 discloses biodegradable CPs by introducing flexible degradable functional groups along the backbone of the CP that can be used for quantitative labeling of mitochondria. Cellular interaction and internalization of CPs are dependent on the chemical structures of both the backbone and side chains of the CPs. CPs with guanidine units (G-CPs) having molecular weights of ˜14,000 g/mol enter live cells quickly, within 10 minutes upon incubation, through the cancer cell membrane.
Conventional method of synthesizing CPs with diverse functional group is tedious and problematic. In addition to intrinsic synthetic challenge of optimizing polymerization conditions for each monomer, the resulting CP with different functional group will have different molecular weight and polydispersity, which will influence their physical and biophysical behaviors. It is therefore desirable to form a nanoparticle or a CP that has attached modified guanidine moieties. These may provide rapid and tailored cellular delivery of anti-MDR siRNA for dramatically enhanced chemotherapy efficiencies that can dramatically impact cancer treatment.